Molecular Imaging of Factor XIII Activity for the Early Detection of Mouse Coronary Microvascular Disease

Figure 1. Optimal PCR parameters were determined based on their effects on primary EC viability and oxidative stress. (A) Cell viability was determined by the MTS reduction assay as described in Materials and Methods. The amount of 490nm absorbance in the presence of different concentrations of rose bengal and various periods of illumination. Each panel is a composite of three experiments. There were statistically significant differences in EC viability between group means (rose bengal concentration, period of green light illumination, and their interaction), assessed by two-way ANOVA (p < 0.01). One-way ANOVA was further used for the effect of different light illumination periods on the same concentration of rose bengal in Fig 1A. One Way ANOVA p-values were reported. The post-hoc Tukey test was finally used to indicate which combinations of rose bengal and green light illumination were significantly different from another groups. (B) Representative immunohistochemical staining of primary ECs for detection of singlet oxygen and ROS (n=4).

Figure 2. Focal endothelium injury was induced by PCR applied to the microvasculature. Representative transmission electron microscopic images (1-2 images/mouse, n = 2 mice/group) documented PCR-induced endothelial injury. 3 h and 24 h post-PCR samples exhibited endothelial injury (A & C) characterized by loss of endothelial integrity, reduced size of ECs, loss of the vesiculo-vacuolar organelle (V), broken tight junctions (TJ), damaged mitochondria (Mito) (B), and disruption of the muscle contractile proteins (Mus) (A) when compared to sham control samples. L: arteriole lumen; BM: basement membrane. Scale bar 1 um.

Figure 3. No flow phenomena indicated focal ischemia in the coronary PCR-induced MVD model. Representative confocal microscopy (n = 4) demonstrated the presence of a “no flow phenomena” following in vivo fluorescein isothiocyanate (FITC)-dextran injection, suggesting coronary arteriole luminal obstruction. (A & C) Fluorescent images of the contra-lateral LV septal wall at (A) 3 and (C) 24 h after PCR-induced microvascular disease (MVD) demonstrated normal perfusion. (B & D) Images were from an ischemia region within the illuminated photothrombotic area. Absent fluorescence of microvessels (white arrows) with intact macrovessels (red arrow) indicated PCR did target the microvasculature. Scale bar is 170 µm. Epi: epicardium; Endo: endocardium.

Figure 4. Coronary microvasculature obstruction with normal epicardial coronaries was induced in the coronary MVD model. (A) Representative coronary angiography (n = 8) from targeted regions demonstrated PCR-induced small arteriole rarefactions without changes in macrovasculature. At the same time, light only or rose bengal only had minimal effects on the macro- and micro-vascular system. Scale bar is 1 mm in Fig 4a for microCT images. Scale bar is 2 mm in Fig 4a for microscopic view. (B) Quantitative analysis further confirmed microvascular rarefaction (<128 um) 24 h post-PCR induction. A two-way ANOVA analysis was used to show there were statistically significant differences (defined as p-value <0.05) in relative vessel number among different interventions (no treatment control, Light, rose bengal, PCR) and among different diameters (size distributions). One-way ANOVA p-values were further used to determine difference within group (different diameter). These p-values (p < 0.01) were reported in Fig 4B. Pairwise comparisons were based on Tukey's test. AP: anteroposterior view of the heart; Lateral: lateral view of the heart; Microscopic: microscopic view.

Figure 5. Global cardiac function and diastolic function were preserved in the coronary MVD model. Cardiac function was assessed by echocardiography in mice before and after MVD induction (n=12). (A) M-mode image of the left ventricle in the parasternal long-axis view, showing depth markers. LV-EDD: left ventricular end-diastolic diameter. LV-ESD: left ventricular end-systolic diameter. (B) Fractional shortening (FS) and ejection fraction were calculated from echocardiographic measurements made during the cardiac cycle. (C) The ratio of peak early diastolic transmitral velocity to annular velocity (E/e′) was calculated and saved as an index of diastolic function. One-way ANOVA p-values are reported for the over-time effect of cardiac function in Fig 5 B & C. p > 0.05.

Figure 6. Oxidative stress and infiltration of inflammatory cells were present in sub-acute or chronic states of the coronary MVD model. (A) 4-4 hydroxy-2-noneal (HNE) is a marker of lipid peroxidation. Nitrotyrosine is a marker for peroxynitrite formation. The control heart without MVD was stained with anti‐4-4 hydroxy-2-noneal (HNE) / anti‐nitrotyrosine antibodies, which served as the negative control. Hearts with PCR-induced MVD (3 h to 3 d) was gradually stained with anti-HNE antibodies in the MVD region, and the amount of nuclei, stained by DAPI, gradually reduced. Hearts with coronary MVD (3 d to 14 d) infarcted regions were intensively stained with anti-nitrotyrosine antibodies, and the amount of nuclei, stained by DAPI, significantly increased, which might indicate inflammatory cell infiltration. (B) Immunohistochemical staining of CD45 and CD68 in the heart tissue from control and MVD-induced mice. CD45 is a marker of leukocytes and CD68 is a marker of phagocytes and other inflammatory cells. In hearts with coronary MVD, the PCR-illuminated region was stained positively with antibodies on 3 d, with staining intensity gradually reduced over time. At the same region, the amount of…

Figure 7. Gene expression demonstrated a pro-thrombotic and impaired fibrinolytic status in the coronary MVD model. Gene expression was investigated for alpha-fibrinogen (A), FXIII a subunit (B), FXIII b subunit (C), tissue plasminogen activator (t-PA) (D), α2-antiplasmin (E), and plasminogen activator inhibitor-1 (PAI-1) (F) expression in MVD regions at multiple time points after MVD induction. Both FXIII a & b subunit expression were significantly up-regulated in MVD compared with normal myocardial regions, thus confirming a role for FXIII in the early formation of MVD, as well as a pro-thrombotic and anti-fibrinolytic status in the setting of PCR-induced MVD in the first 24 h. Values are mean ± SEM, n=4. One-way analysis of variance (ANOVA) analysis was used to test whether there was a statistical significant effect of lesion aging on gene expression of key molecules. Student t test was used for each time point and these p-values are reported. ** p < 0.01; * p < 0.05

Figure 8. Both ex vivo gamma well counting 24 h-post MVD induction and in vivo serial microSPECT/CT Imaging confirmed elevated FXIII activity in in the coronary MVD model. (A) Radioactivity of FXIII in the PCR-induced MVD region increased more than 20-folds at 24 h, when compared to light only and rose bengal only groups. FXIII activity also increased approximately 2-fold in the normal region compared to the same region exposed to light only or rose bengal only. (n = 8) A one-way ANOVA analysis was used to test whether there was a statistically significant effect of intervention on NC100668 biodistribution in specific organs. These p-values (p < 0.01) were reported in Fig 8A. A post-hoc Tukey-test was further used, indicating that activity in the PCR group was significantly higher than those of light only and rose bengal only groups (p < 0.01). There were no activity differences between light only and rose bengal only groups. (B) Retention of 99mTc-NC100668 was dramatically increased in the kidney, liver, and spleen, indicating the clearance is comparable among the three experimental groups (PCR, light only, and rose bengal only). (C) Representative in vivo serial 99mTc-NC100668…